Phosphatidic acid (PA) synthesised via the glycerol-3-phosphate or the dihydroxyacetone phosphate pathway is an important intermediate in the biosynthesis of glycerophospholipids and triacylglycerols [Athenstaedt and Daum, Eur. J. Biochem. 266: 1-16 (1999)]. In addition, because PA can be produced from hydrolysis of phosphatidylcholine (PC) by phospholipase D (PLD), it is recently attracting considerable interest as a potential second messenger. This hypothesis is based on the observation that many agonists cause PLD-activation (and thus PA formation) in a variety of cell types and tissues to regulate many cellular pathways including secretion, respiratory burst, calcium influx, mitosis, etc [Exton, Biochimica et Biophysica Acta 1439: 121-133 (1999)]. If PA is indeed a second messenger, it would be expected to interact specifically with a certain class of cellular proteins. However, the identification of such proteins has not been achieved to date in a systematic approach, and would be extremely complicated by conventional methods for the following reasons:                1. PA is unstable in cells since it is rapidly dephosphorylated to give diacylglycerol [Hodgin et al. TIBS 23: 200-204 (1998)].        2. Free PA (both synthetic or from biological origin) is difficult to handle due to its poor solublity in aqueous media. Extensive sonication to obtain liposomes is hence required. (Pure naturally-occurring or synthetic PA is difficult to solubilise in vitro, requiring either organic solvents or extensive sonication to produce liposomes).        3. Even if PA were reconstituted into liposomes, it would be impossible to identify detergent-extracted PA-binding proteins (vide infra) since the detergent used would destroy the integrity of the liposome structure.        